anti bcl x l 54h6 rabbit mab  (Cell Signaling Technology Inc)

Journal: Cell Death & Disease

Article Title: Contribution of A1 to macrophage survival in cooperation with MCL-1 and BCL-X L in a murine cell model of myeloid differentiation

doi: 10.1038/s41419-024-07064-z

Figure Lengend Snippet: A1 was knocked out in Hoxb8 GM-SCF macrophage progenitors by lentiviral CRISPR/Cas9 using two different sgRNAs (A1g7 and A1g12) targeting all isoforms (A1g7) or A1a, c and d (A1g12). An irrelevant sgRNA sequence directed against EGFP served as control (CTRL). a – d Hoxb8 macrophage progenitors were treated for 24 h with the following inhibitors targeting anti-apoptotic BCL-2-family proteins: ABT-737 ( a , specific for BCL-X L and BCL-2), S63845 ( b , specific for MCL-1), ABT-199 ( c , specific for BCL-2) or A-1155463 ( d , specific for BCL-X L ) at the concentrations (µM) indicated. e , f Hoxb8 macrophage progenitors were treated as above with single or combined inhibitors as indicated. DMSO served as solvent control. Cell death was assessed by AnnexinV/PI staining and flow cytometry (FACS Calibur). Shown are the percentage of live cells (AnnexinV and PI-negative). Data are means/SEM of 3–7 independent experiments. Statistical analysis was done by 2-way ANOVA. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns, non-significant.

Article Snippet: Membranes were probed with antibodies against murine A1 (clone 6D6, gift from Marco Herold, WEHI, Melbourne, Australia), MCL-1 (#600-401-394, Rockland), BCL-2 (clone 3F11, BD), BCL-X L (clone 54H6, Cell Signaling), BCL-w (clone 31H4, Cell Signaling) BAX (#2772, Cell Signaling), BAK (#06-536, Milipore), BIM (clone C34C5, Cell Signaling) NOXA (#ab36833, Abcam), PUMA (clone E1S7A, Cell Signaling) and GAPDH (#MAB384, Merck/Millipore).

Techniques: CRISPR, Sequencing, Control, Solvent, Staining, Flow Cytometry

Journal: Cell Death & Disease

Article Title: Contribution of A1 to macrophage survival in cooperation with MCL-1 and BCL-X L in a murine cell model of myeloid differentiation

doi: 10.1038/s41419-024-07064-z

Figure Lengend Snippet: A1-KO or CTRL GM-CSF Hoxb8 cells (A1g7, A1g12 or CTRL) were differentiated into macrophages for 7 days by oestrogen withdrawal. a – d Differentiated macrophages were treated for 4 h with the following inhibitors targeting anti-apoptotic BCL-2-family proteins: ABT-737 ( a , specific for BCL-X L and BCL-2), S63845 ( b , specific for MCL-1), ABT-199 ( c , specific for BCL-2) or A-1155463 ( d , specific for BCL-X L ) at the concentrations (µM) indicated. e – h Differentiated macrophages were treated as above with single or combined inhibitors as indicated. DMSO served as solvent control. Cell death was assessed by active-Caspase-3 staining and flow cytometry (FACS Calibur). Data are means/SEM of 3–7 independent experiments. Statistical analysis was done by 2-way ANOVA. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns, non-significant.

Article Snippet: Membranes were probed with antibodies against murine A1 (clone 6D6, gift from Marco Herold, WEHI, Melbourne, Australia), MCL-1 (#600-401-394, Rockland), BCL-2 (clone 3F11, BD), BCL-X L (clone 54H6, Cell Signaling), BCL-w (clone 31H4, Cell Signaling) BAX (#2772, Cell Signaling), BAK (#06-536, Milipore), BIM (clone C34C5, Cell Signaling) NOXA (#ab36833, Abcam), PUMA (clone E1S7A, Cell Signaling) and GAPDH (#MAB384, Merck/Millipore).

Techniques: Solvent, Control, Staining, Flow Cytometry

Journal: Cell Death & Disease

Article Title: Contribution of A1 to macrophage survival in cooperation with MCL-1 and BCL-X L in a murine cell model of myeloid differentiation

doi: 10.1038/s41419-024-07064-z

Figure Lengend Snippet: a , b A1-KO and WT CTRL GM-CSF progenitors were co-treated with LPS (1 µg/ml) and inhibitors against BCL-X L /BCL-2 (ABT-737) or MCL-1 (S63845) at the concentrations (µM) indicated. DMSO served as solvent control. Viability and apoptotic cell death were assessed after 4 h by AnnexinV/PI staining ( a , shown are the percentage of live, AnnexinV and PI-negative cells) and staining against active caspase-3 ( b ) followed by flow cytometry analysis (FACS Calibur). Data are means/SEM of 3–7 independent experiments. Statistical analysis was done by 2-way ANOVA. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns, non-significant.

Article Snippet: Membranes were probed with antibodies against murine A1 (clone 6D6, gift from Marco Herold, WEHI, Melbourne, Australia), MCL-1 (#600-401-394, Rockland), BCL-2 (clone 3F11, BD), BCL-X L (clone 54H6, Cell Signaling), BCL-w (clone 31H4, Cell Signaling) BAX (#2772, Cell Signaling), BAK (#06-536, Milipore), BIM (clone C34C5, Cell Signaling) NOXA (#ab36833, Abcam), PUMA (clone E1S7A, Cell Signaling) and GAPDH (#MAB384, Merck/Millipore).

Techniques: Solvent, Control, Staining, Flow Cytometry

Journal: Cell Death & Disease

Article Title: Contribution of A1 to macrophage survival in cooperation with MCL-1 and BCL-X L in a murine cell model of myeloid differentiation

doi: 10.1038/s41419-024-07064-z

Figure Lengend Snippet: a – c Day 7 differentiated Hoxb8 macrophages were co-treated with LPS and single or combined inhibitors against BCL-X L /BCL-2 (ABT-737), MCL-1 (S63845), BCL-2 (ABT-199) or BCL-X L (A-1155463) at the concentrations (µM) indicated for 4 h. DMSO served as solvent control. Viability and apoptotic cell death were assessed by AnnexinV/PI staining ( a ) and staining against active caspase-3 ( b , c ), followed by flow cytometry (FACS Calibur). Data are means/SEM of 3 independent experiments. Statistical analysis was done by 2-way ANOVA. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns, non-significant.

Article Snippet: Membranes were probed with antibodies against murine A1 (clone 6D6, gift from Marco Herold, WEHI, Melbourne, Australia), MCL-1 (#600-401-394, Rockland), BCL-2 (clone 3F11, BD), BCL-X L (clone 54H6, Cell Signaling), BCL-w (clone 31H4, Cell Signaling) BAX (#2772, Cell Signaling), BAK (#06-536, Milipore), BIM (clone C34C5, Cell Signaling) NOXA (#ab36833, Abcam), PUMA (clone E1S7A, Cell Signaling) and GAPDH (#MAB384, Merck/Millipore).

Techniques: Solvent, Control, Staining, Flow Cytometry

Journal: Genes & Genomics

Article Title: Negative regulation of HDAC3 transcription by histone acetyltransferase TIP60 in colon cancer

doi: 10.1007/s13258-024-01524-8

Figure Lengend Snippet: HDAC3 suppression by TIP60 regulated the proliferation and apoptosis of HCT116 cells. A (Left) Representative images of colony formation assay of HCT116 cells in each condition. (Right) The bar graph represents the result of colony formation assay. Data are shown as the mean ± SEM (n = 3). P -values were calculated using ANOVA followed by Dunnett’s multiple comparison test. * P < 0.05 and n.s., not significant. B The bar graph represents RT-qPCR analysis of the mRNA levels of apoptosis-related genes in TIP60 overexpressing HCT116 cells. Data are shown as the mean ± SEM (n = 3). P -values were calculated using paired two-tailed Student’s t-tests. * P < 0.05. C The proportion of apoptotic cells were analyzed by flow cytometry. Transfected cells were treated with 5 mM of hydroxyurea (HU) for 32 h to induce apoptosis and stained with propidium iodide (PI) and FITC-Annexin V after trypsinization. D Western blot analysis to evaluate apoptosis-related protein levels including PARP1 and Bcl-X L in each condition. Asterisk marks (*) indicate cleaved form of PARP1

Article Snippet: After transfer, the membranes were incubated at 4℃ overnight with the primary antibodies against TIP60 (sc-166323; dilution 1:5,000), β-actin (sc-47778, dilution 1:1,000) and Bcl-X L (sc-7195, dilution 1:500) from Santa Cruz Biotechnology (Dallas, TX, USA); HDAC2 (ab12169, dilution 1:5,000), HDAC3 (ab32369; dilution 1:5,000), γ-H2A.X (ab2893, dilution 1:5,000) and PARP1 (ab32138, dilution 1:5,000) from Abcam (Cambridge, UK); HDAC4 (7628S; dilution 1:5,000) from Cell Signaling Technology (Danvers, MA, USA); FLAG (F3165, dilution 1:5,000), HDAC1 (06–720; dilution 1:5,000) and Vinculin (V9131, dilution 1:2,500) from Sigma-Aldrich (Burlington, MA, USA).

Techniques: Colony Assay, Comparison, Quantitative RT-PCR, Two Tailed Test, Flow Cytometry, Transfection, Staining, Western Blot